Preferred drug screening method of ion channels include the standard high throughput screens (HTS) using mixtures of test compounds and biological reagents along with indicator compound loaded into cells in arrays of wells, usually in standard microtiter plates with 96 or 384 wells, and measuring the signal from each well, either fluorescence emission, intracellular pH, optical density, radioactivity, etc.
The GABAA receptor is an attractive target for the development of new drugs. The GABAA receptor, however, is a chloride channel, rendering it incompatible with conventional HTS methods. Moreover, most cells capable of functionally expressing GABAA receptors do not maintain a sufficient chloride gradient across their membranes. For validating these targets, cost and time-consuming electrophysiological methods are still the preferred methods.
There is a strong felt need in the art to make these receptors the target for more efficient and inexpensive high throughput drug screening methods.
Wang et al. [Wang Chih-Tien et al: Cation Permeability and Cation-Anion Interactions in a Mutant GABA-Gated Chloride Channel from Drosophila; Biophys. J. 1999 77 691-700] describe an insect GABA receptor that has been made cation permeable by mutation in the near-M2 region. This receptor, however, does not contain modulator sites affected by drugs, and is not suited for high throughput drug screening.
Bertrand et al. [Bertrand D, Galzi J L, Devillers-Thieiy A, Bertrand S & Changeux J P: Mutations at two distinct sites within the channel domain M2 alter calcium permeability of neuronal α7 nicotinic receptor; Proc. Natl. Acad. Sci. USA 1993 90 6971-6975] describe how mutations at two distinct sites within its TM2 domain alter the calcium permeability of a nicotinic α7 receptor.
Keramidas et al. [Keramidas A, Moorhouse A J, French C R, Schofield P R & Barry P H. M2 Pore Mutations Convert the Glycine Receptor Channel from Being Anion- to Cation-Selective; Biophys. J. 2000 78 247-259] describe how three mutations carried out in the M2 transmembrane domain of the chloride-conducting α1 homomeric glycine receptor alter the receptor from being anion- to cation-selective.
A cation-conducting GABAA receptor suited for high throughput drug screening has never been disclosed.